Hemolytic syndromes including sickle cell disease are devastating illnesses that affect over 100,000 people in the United States. Each of these patients suffers a broad spectrum of pulmonary and systemic vascular complications caused by red blood cell hemolysis, high plasma levels of cell free hemoglobin (Hb), endothelial cell dysfunction, and tissue hypoxia. WHO Class 5 pulmonary hypertension (PH), a devastating and fatal disease, is significantly increased in this population, without a clear mechanistic explanation. This grant application and studies outlined herein focus on mechanisms and attenuation of class 5 PH, secondary to hemolysis. Toll-like receptors (TLR) are pattern receptors that regulate innate inflammation. It has been postulated that their activation contributes to the development of PH. Hb is a pro-oxidant with the capability to damage mitochondria and subsequently activate endothelial and smooth muscle cell TLR9. We there- fore hypothesize that Hb-induced oxidative stress leads to mitochondrial damage and activation of TLR9 and NF-?B signaling through the MyD88 protein contributing to PH. We propose a three part mechanism by which this occurs: (1) HbFe2+ extravasates from the plasma into the intima, media and adventitia; (2) Once in the extravascular (i.e.tissue) compartment, HbFe2+ is oxidized to HbFe3+;and (3) Hb redox reactions and release of heme and non-heme iron within the tissue layers causes oxidative stress resulting in vascular damage and release of Danger Associated Molecular Patterns (DAMPS) in the form of DNA, which activate TLR9. Three specific aims are proposed: Aim 1 will test the hypothesis that extravasated Hb and subsequent break- down into heme or non-heme iron causes tissue damage from increased oxidative stress is the root cause in Hb- mediated PH in vivo. Aim 2 will test the hypothesis that Hb or heme-induced ROS and lipid peroxidation activate TLR9 from non-compartmentalized DNA due to either cell death acting on adjacent cells or from mtDNA that es- capes mitophagy and auto-activates its own cellular TLR9. Aim 3 will test: (a) the hypothesis that TLR9 loss- of-function either globally or specifically within endothelial or smooth muscle cell will attenuate Hb-mediated PH in vivo; (b) the hypothesis that Hb-mediated TLR9 signaling occurs through the canonical NF-?B vs.a p38 MAPK or AMPK pathway in both the endothelial and smooth muscle cell in vitro; and (c) the hypothesis that Hb-mediated TLR9 signaling induces an EC phenotype that evokes SMC proliferation in vitro. An in-depth understanding of these relationships will allow us to identify new therapies against Hb's contribution to vascular diseases, which can be used to enhance and extend the lives of people vulner- able to class 5 PH, secondary to hemolysis.